The novel hyaluronic acid granular hydrogel attenuates osteoarthritis progression by inhibiting the TLR‐2/NF‐κB signaling pathway through suppressing cellular senescence

Abstract In patients with mild osteoarthritis (OA), two to four monthly injections are required for 6 months due to the degradation of hyaluronic acid (HA) by peroxidative cleavage and hyaluronidase. However, frequent injections may lead to local infection and also cause inconvenience to patients during the COVID‐19 pandemic. Herein, we developed a novel HA granular hydrogel (n‐HA) with improved degradation resistance. The chemical structure, injectable capability, morphology, rheological properties, biodegradability, and cytocompatibility of the n‐HA were investigated. In addition, the effects of the n‐HA on the senescence‐associated inflammatory responses were studied via flow cytometry, cytochemical staining, Real time quantitative polymerase chain reaction (RT‐qPCR), and western blot analysis. Importantly, the treatment outcome of the n‐HA with one single injection relative to the commercial HA product with four consecutive injections within one treatment course in an OA mouse model underwent anterior cruciate ligament transection (ACLT) was systematically evaluated. Our developed n‐HA exhibited a perfect unification of high crosslink density, good injectability, excellent resistance to enzymatic hydrolysis, satisfactory biocompatibility, and anti‐inflammatory responses through a series of in vitro studies. Compared to the commercial HA product with four consecutive injections, a single injection of n‐HA contributed to equivalent treatment outcomes in an OA mouse model in terms of histological analysis, radiographic, immunohistological, and molecular analysis results. Furthermore, the amelioration effect of the n‐HA on OA development was partially ascribed to the attenuation of chondrocyte senescence, thereby leading to inhibition of TLR‐2 expression and then blockade of NF‐κB activation. Collectively, the n‐HA may be a promising therapeutic alternative to current commercial HA products for OA treatment.


| INTRODUCTION
Osteoarthritis (OA) is a serious chronic degenerative disease characterized by articular cartilage loss, synovitis, subchondral bone sclerosis, and osteophyte, which is the main cause of joint pain and disability. [1][2][3][4][5][6] The progression of OA may ultimately lead to keen arthroplasty surgery to restore joint function. 7,8 Although there are no effective disease-modifying therapies available to delay or prevent knee replacement, intra-articular therapies such as corticosteroids (CS), nonsteroidal anti-inflammatory drugs (NSAIDs), and hyaluronic acid (HA) have been commonly used in clinical trials to mitigate cartilage destruction and knee pain. [9][10][11] However, the prolonged use of CS may result in chondrocyte apoptosis while the frequent injection of NSAIDs may cause adverse effects on liver and kidney functions. 12 Compared to the healthy group, the OA patients have less HA with lower quality in their knee joint cavities, thereby leading to higher friction due to the reduced viscoelasticity of synovial fluid. 13 Therefore, injection of HA viscosupplements is one of the most recommended strategies for pain relief in patients suffering from mild-to-moderate OA in clinics. 14 Because of the existence of hyaluronidase and reactive oxygen species (ROS) in human body, the routine therapeutic mode of current commercial HA products requires four injections monthly for effective relief of knee pain, thus easily causing inconvenience for patients especially in the COVID-19 pandemic and also pain at the injection site. [15][16][17] In addition, frequent injection may increase the chance of joint infection. 18 To improve the enzymatic or ROS resistance of HA and reduce the risk of infection from multiple injections, an attractive strategy is to cross-link HA through specific chemical reactions to generate HA hydrogel systems. [19][20][21][22] For example, chemical reagents, such as divinylsulfone (DVS), glycidyl methacylate (GMA), and butanediol diglycidyl ether (BDDE), are commonly adopted to attain chemically crosslinked HA with similar molecular weight to endogenous HA. 23,24 However, clinical concerns have been raised on the biosafety of these cross-linking agents. For example, the residual cross-linking agent in joints may cause severe inflammatory symptoms including joint swelling and redness, local pressure, skin rash, vomiting, and fever. 18 Moreover, since the HA cross-linking process may impair the injectability of HA materials, how to balance the injectability and resistance to enzymatic or ROS degradation of HAbased hydrogels is thus another critical concern.
Granular hydrogels, also known as densely packed or jammed hydrogel microparticles, have recently contributed significantly to advances in material chemistry and tissue engineering because of their unique properties including inherent porosity, shear-thinning, selfhealing, modular nature, and so on. 1,4,25 Inspired by this, we reported a novel BDDE-crosslinked HA granular hydrogel (n-HA) for the first time as a promising therapeutic alternative to current commercial HA products for OA treatment. First, the biocompatibility and bioefficacy of n-HA were systematically investigated to test its potential as a viscous supplement for OA patients through a series of in vitro and in vivo experiments. Then, the potential mechanism behind n-HA attenuating OA development was uncovered. We found that n-HA can block TLR-2/NF-κB signaling pathway by inhibiting cellular senescence, thereby exerting anti-inflammatory and cartilage protective effects. This work may layout the foundation for fundamental and translational research on potential therapies for OA. After the addition of 0.5, 0.8, 1, or 1.5 vol% 1,4-butanediol diglycidyl ether (BDDE: ρ = 1.1 g/ml, purity ≥ 95% from Aladdin Co., Ltd., Shanghai, China) into the HA solution, the mixture was then emulsified with the fluorinert™ FC-40 oil containing 2 wt% Pico-Surf™ as the oil phase. Afterwards, the mixture was kept at 40 C for 4-6 h to obtain the HA hydrogel microparticles. The hydrogel microparticles were further screened by different mesh screens to collect products with sizes ranging from 100 to 150 μm. Then, the hydrogel microparticles were purified with 2-(perfluorohexyl)ethyl alcohol and saline.
n-HA was finally obtained at a concentration of 10 mg/ml by centrifugation to remove excess saline and used for the following experiments. The nomenclature of the various n-HAs discussed in this study was achieved using the volume fraction of BBDE applied in the synthetic steps as the n-HA prefix. For example, 0.5% n-HA corresponds to the n-HA sample composed of hydrogel microparticles synthesized using 0.5 vol% BDDE.

| Morphology characterization of the granular gel after injection
Morphology characterization of n-HA after injection was carried out by an inverted microscope (BDS400; Conptec, China) and a confocal microscopy (LSM710; Zeiss, Germany). For inverted microscopy characterization, n-HA was directly dispersed in saline and observed.
Moreover, n-HA was dispersed in saline containing tetramethylrhodamine isothiocyanate-dextran (20 kDa, SIGMA) to detect the interstitial space using confocal microscopy.
2.2.2 | Nuclear magnetic resonance and Fouriertransform infrared spectroscopy 1 H NMR spectra (400 MHz) of HA, 0.5% n-HA, 0.8% n-HA, 1% n-HA, and 1.5% n-HA in D 2 O were analyzed by a Bruker ACF-400 spectrometer. Since n-HA is composed of crosslinked hydrogel microparticles, which are poorly soluble in water, n-HA was completely digested using hyaluronidase (Aladdin, 300 U/ml). Afterward, the supernatant was collected through centrifuge and then lyophilized and redispersed in D 2 O for 1 H NMR analysis. Fourier-transform infrared (FTIR) of HA, 0.5% n-HA, 0.8% n-HA, 1% n-HA, and 1.5% n-HA were recorded employing a Nicolet 6700 spectrometer (Thermo, USA) from the wave numbers 4000 to 400 cm À1 .

| Injectable capacity and morphology of n-HA
The n-HA was aspirated into a syringe with 25 G needle to test their injectable capacity. The cross-sectional morphology of freeze-dried n-HA was examined by scanning electron microscopy (SEM) (Quanta 400F; Philips, Netherlands).

| Rheological properties and biodegradability
The rheological properties of n-HA and the commercial HA product (ARTZ ® , Japan, 10 mg/ml) were measured using the Haake Mars 40 Rheometer with parallel plate geometry (8 mm flat plate). All rheological tests were performed at 25 C. Specifically, amplitude sweep tests were carried out with oscillation strain amplitudes of 0.1%-1000% at a constant angular frequency of 1 Hz. The frequency sweep tests were performed in the angular frequency range of 0.1-100 rad/s at a constant strain amplitude of 1%.
Enzymatic degradation experiments of n-HA and ARTZ ® were performed according to previously published protocols. 26,27 In brief, 2 g of n-HA or ARTZ ® was added to 2 ml solution (30 mM citric acid, 150 mM Na 2 HPO 4 , 150 mM NaCl, pH 6.3) supplemented with 300 U/ml hyaluronidase (HAase) at 37 C. The 100 μl of the supernatant was taken at different time intervals, and the content of uronic acid was determined by the carbazole method to determine the degradation rate of the sample. 26 ROS-induced degradation rate of n-HA was determined using a gravimetric method. Specifically, newly formed 0.5%, 0.8%, and 1.

| Cell cytotoxicity
The primary chondrocytes were seeded in a 96-well plate at a density of 5 Â 10 3 cells/well and cultured overnight prior to the addition of a series levels (0.2-5 mg/ml) of n-HA or ARTZ ® . After 24 h, cytotoxicity assessment was conducted using the Cell Counting Kit-8 (CCK-8, GLPBIO, GK10001, USA) assay.
In addition, the cytocompatibility of n-HA and ARTZ ® was assessed by live and dead staining assay. In a nutshell, the isolated chondrocytes at passage one or two were seeded in a 48-well plate at a density of 1 Â 10 4 cells/well and cultured overnight prior to the addition of 1 mg/ml of n-HA or ARTZ ® . After 1, 2, and 3 days, the viability of the cells was analyzed using the Live/Dead cell-mediated cytotoxicity kit (US Everbright Inc.).

| Construction of cell inflammation model and cell senescence model
In order to construct a cellular inflammation model, we used TNF-α (Affinity Biosciences, BF0170-100) with 20 ng/ml to culture chondrocytes for 24 h ( Figure S1). In addition, normal chondrocytes were stimulated with TNF-α (10 ng/ml) for 48 h to induce chondrocyte senescence, thereby constructing an in vitro cell senescence model. 28

| Phalloidin staining
The chondrocytes were seeded in laser confocal dish at a density of 2 Â 10 4 cells/dish and cultured overnight. The 1 mg/ml of n-HA or ARTZ ® was then added in the presence of TNF-α with 20 ng/ml. After 24 h, cells were incubated with red phalloidin fluorescent working solution (Abcam, USA) for 60 min according to the method described in the instruction and then incubated with DAPI dye for 5 min. Confocal laser scanning microscopy (CLSM) (FV3000; Olympus) was used to observe cell morphology. and then stained with Annexin V-FITC for 10 min at room temperature, followed by PI staining for another 5 min. After that, the cells were analyzed by flow cytometry (BD, USA). The number of events recorded in the sample was 10,000 cells.

| Senescence-associated-β-galactosidase staining assay
Normal chondrocytes were stimulated with TNF-α (10 ng/ml) for 48 h to induce chondrocyte senescence, thereby constructing an in vitro cell senescence model. Cellular senescence was then measured using a senescence-associated-β-galactosidase (SA-β-Gal) assay kit (Beyotime, China). The senescent cells were seeded in a 96-well plate at a density of 5 Â 10 3 cells/well and cultured overnight. The 1 mg/ml of n-HA or ARTZ ® was then added into the cell culture medium. After 24 h, the cells were washed twice with PBS after aspiration of the culture medium. Afterward, the cells were fixed by the SA-β-Gal staining fixative for 15 min at room temperature. After that, the cells were washed with PBS for three times. Finally, the staining working solution was added for incubation overnight at 37 C in an incubator. The number of SA-β-Gal positive cells from three random fields was recorded for calculation of the average counts.  Table S1.

| Animal model
All the operations were performed with the approval by the local Animal Care and Use Committee (SYSU-IACUC-2020-000328). Thirty 8-12 weeks male C57BL/6 mice were purchased and housed in a specific-pathogen-free animal facility. Mice were divided into five groups randomly. Destabilization of the medial meniscus (DMM) surgery was conducted in mice to induce OA model. After 2 weeks, according to treatment instruction in clinics, a total volume of 10 μl of saline, n-HA (10 mg/ml), or ARTZ ® (10 mg/ml) was intra-articularly injected into the mice with a microinjector. The saline and ARTZ ® were administrated for 4 consecutive weekly intra-articular injections while n-HA was conducted for only one single injection during a course of treatment.

| Micro-CT analysis
The potential changes of subchondral trabecular bone microarchitecture and mineral density in the region of interest (ROI) beneath subchondral cortical bone were measured by micro-CT. Briefly, the knee joint of the mouse was isolated after euthanasia, and the surrounding soft tissue was removed. After fixation with 4% paraformaldehyde solution for 24 h, the knee joints were scanned using a micro-CT system (Scano Medical μCT 100, Switzerland) with the scanning parameters set as 55 kV, 200 μA, and 10 μm in spatial resolution.

| Histological and immunohistochemical analysis
The tissue sections were cut with a thickness of 4 mm along the coronal plane. Cartilage injury was assessed by Safrani O/Fast Green staining and toluidine blue staining. The OARSI OA histopathology assessment system was chosen to assess the cartilage degeneration severity. The articular cartilage thickness was measured by image J, while the proteoglycan content was scored based on the reported modified Mankin scoring system. 29 For immunohistochemical staining, the sectioned tissue was treated by high temperature in buffers for antigen retrieval prior to incubation in 3% H 2 O 2 for 15 min to quench endogenous catalase. The blocked sections with serum were then incubated with primary antibodies recognizing TLR-2 antibody (Abcam, USA), P16 INK4a antibody (Bioss), COL2A1 antibody (Bioss), and MMP13 antibody (Bioss), respectively.
After being washed three times with PBS and incubated with goatanti-rabbit HRP-conjugated secondary antibody for 1 h at 37 C, an immunohistochemical staining signal was developed with DAB substrate system. Image J software (National Institutes of Health, MD, USA) was used to quantify and analyze IHC-positively stained cells.

| Statistical analysis
Data are presented as mean ± standard deviation (mean ± S.D). Differences between two groups were statistically analyzed by unpaired, two-tailed Student's t test while analysis of variance (ANOVA) with Dunnett post hoc test was used for the comparison of data in more than two groups of variables. The level of significance was set at *p < 0.05, **p < 0.01, ***p < 0.001. All statistical analyses were  Figure S2). Accordingly, the MoD of 0.5%, 0.8%, 1%, and 1.5% n-HA samples was calculated to be 11%, 14.75%, 19%, and 25.75%, respectively (Table S2).
FTIR spectra of lyophilized HA and n-HA samples were depicted in Figure 2b, which showed similar characteristic peaks for all the samples. Afterward, the manual injectability of n-HAs was evaluated. As  Figure S3).
Then, the degradation behavior of n-HA and ARTZ ® by peroxidative cleavage or hyaluronidase was evaluated. As shown in Figure 2f, the complete degradation time of ARTZ ® was 4 days, while that of 1% n-HA was 7 days, indicating that BDDE cross-linking endows n-HA with good hyaluronidase resistance. Similar results were also noticed for the degradation of n-HA by peroxidative cleavage ( Figure S4). Consequently, compared to ARTZ ® , 1% n-HA showed improved resistance to degradation and was therefore selected in the following in vitro and in vivo tests.

| n-HA inhibits inflammation-induced chondrocyte apoptosis and necrosis without causing cytotoxicity
The cytocompatibility of n-HA was evaluated by using primary chondrocytes via cell viability and live/dead staining assay. First, we pre-  (Figure 6b,c). No matter n-HA or ARTZ ® injection significantly prevented cartilage loss and degeneration (Figure 6d,e). OARSI cartilage scoring evaluation has been widely used for quantitative assessment of OA severity.
As shown in Figure 6f, compared with the OA group without treatment, n-HA or ARTZ ® intervention significantly reduced OARSI scores. Among all the treatment groups, n-HA exhibited best performance in reduction of OARSI scores although there was no significant difference in OARSI scores between n-HA and ARTZ ® groups.
Afterward, the quantitative analysis of subchondral bone sclerosis of tibiae in the OA model mice with various interventions was performed by micro-CT technique. As compared to the sham group, both of the OA group and the saline group showed significantly higher plate thickness (Figure 7a,b), bone mineral density (BMD) (Figure 7c 37 Most recently, a n-HA product (Durolane) with very high molecular weight over 100,000 kDa has been tried to treat mild to moderate knee and hip OA patients, revealing beneficial while not significant improved effects. 38 The controversial clinical outcomes may be caused by injection dose, HA crosslinking degree, OA stages, and criteria. Although HA has been widely used in clinics, the repair mechanism was still lacking. Recent work has found that HA can inhibit synovial inflammation through modulation of GRP78/NF-κB signaling pathway, contributing to significant reduction of IL-6 and PGE2 levels. 30 In addition, HA is negatively correlated with the production of IL-1β-stimulated MMPs and free radicals, 39 thereby mitigating chondrocyte apoptosis and articular cartilage degeneration.

| DISCUSSION
Interestingly, HA even showed protective role in tissue-engineered cartilage in the presence TNF-α via upregulation of VEGFA and ANKRD37 genes. 40 Recently, cellular senescence have been viewed as a critical factor influencing cartilage loss and joint inflammation. 30 As the production of pro-inflammatory molecules and degenerative enzymes was closely associated with SASP caused by cellular senescence, so local clearance of senescent cells was a promising therapeutic target for attenuation of OA development. 41 Encouragingly, our developed n-HA granular hydrogel exhibited the potential in suppression of cellular senescence, contributing to reduced SASP production and thereby inhibiting TLR-2 levels via a pro-inflammatory cytokine Notably, no extra cytotoxicity or organ toxicity was caused after the use of n-HA granular hydrogel with the equivalent dose to the commercial HA product ( Figure S1).
However, there were some limitations to this study. First, various mild to moderate OA models mimicking different clinical phenotypes in OA patients should be considered to validate the bio-efficacy of our developed n-HA granular hydrogel. Then, a longer treatment course may be required for verification of superiority, equivalence or non-inferiority of n-HA relative to its commercial counterpart in OA models prior to the potential clinical trials. Although our proof study revealed the advantage of n-HA with improved resistance to enzymatic hydrolysis, there are still some concerns about the potential phagocytosis of such granular microgels by macrophages. Therefore, it is important to track the fate of n-HA granular microgels in vivo through signal labeled technology.

| CONCLUSION
We synthesized a n-HA granular hydrogel for the intervention treat-